The aberrant expression in epithelial cells of the mesenchymal isoform of FGFR2 controls the negative crosstalk between EMT and autophagy

Signalling of the epithelial splicing variant of fibroblast growth factor receptor 2 (FGFR2b) triggers both differentiation and autophagy, while the aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells induces impaired differentiation, inhibition of autophagy as well as the induction of the epithelial-mesenchymal transition (EMT). In light of the widely proposed negative loop linking autophagy and EMT in the early steps of carcinogenesis, here we investigated the possible involvement of FGFR2c aberrant expression and signalling in orchestrating this crosstalk in human keratinocytes. Biochemical, molecular, quantitative immunofluorescence analysis and in vitro invasion assays, coupled to the use of specific substrate inhibitors and transient or stable silencing approaches, showed that AKT/MTOR and PKCε are the two hub signalling pathways, downstream FGFR2c, intersecting with each other in the control of both the inhibition of autophagy and the induction of EMT and invasive behaviour. These results indicate that the expression of FGFR2c, possibly resulting from FGFR2 isoform switch, could represent a key upstream event responsible for the establishment of a negative interplay between autophagy and EMT, which contributes to the assessment of a pathological oncogenic profile in epithelial cells

Ire1 alpha/xbp1 axis sustains primary effusion lymphoma cell survival by promoting cytokine release and stat3 activation

Primary Effusion Lymphoma (PEL) is a highly aggressive B cell lymphoma associated with Kaposi’s Sarcoma-associated Herpesvirus (KSHV). It is characterized by a high level of basal Endoplasmic Reticulum (ER) stress, Unfolded Protein Response (UPR) activation and constitutive phosphorylation of oncogenic pathways such as the Signal Transducer and activator of Transcription (STAT3). In this study, we found that the inositol requiring kinase (IRE) 1alpha/X-box binding protein (XBP1) axis of UPR plays a key role in the survival of PEL cells, while double stranded RNA-activated protein kinase-like ER kinase (PERK) and activating transcription factor (ATF) 6 slightly influence it, in correlation with the capacity of the IRE1alpha/XBP1 axis to induce the release of interleukin (IL)-6, IL-10 and Vascular-Endothelial Growth Factor (VEGF). Moreover, we found that IRE1alpha/XBP1 inhibition reduced STAT3 Tyr705 phosphorylation and induced a pro-survival autophagy in PEL cells. In conclusion, this study suggests that targeting the IRE1alpha/XBP1 axis represents a promising strategy against PEL cells and that the cytotoxic effect of this treatment may be potentiated by autophagy inhibition

Uncoupling GP1 and GP2 expression in the Lassa virus glycoprotein complex: implications for GP1 ectodomain shedding

<p>Abstract</p> <p>Background</p> <p>Sera from convalescent Lassa fever patients often contains antibodies to Lassa virus (LASV) glycoprotein 1 (GP1), and glycoprotein 2 (GP2); Immunization of non-human primates with viral vectors expressing the arenaviral glycoprotein complex (GPC) confers full protective immunity against a lethal challenge with LASV. Thus, the development of native or quasi native recombinant LASV GP1 and GP2 as soluble, uncoupled proteins will improve current diagnostics, treatment, and prevention of Lassa fever. To this end, mammalian expression systems were engineered for production and purification of secreted forms of soluble LASV GP1 and GP2 proteins.</p> <p>Results</p> <p>Determinants for mammalian cell expression of secreted uncoupled Lassa virus (LASV) glycoprotein 1 (GP1) and glycoprotein 2 (GP2) were established. Soluble GP1 was generated using either the native glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain was deleted, the intracellular domain (IC) was fused to the ectodomain, and the gene was co-expressed with a complete GP1 gene in <it>cis</it>; (2) the TM and IC domains were deleted and GP1 was co-expressed in <it>cis</it>; (3) expression of GP1 was driven by the native GPC SP. These data implicate GP1 as a chaperone for processing and shuttling GP2 to the cell surface. The soluble forms of GP1 and GP2 generated through these studies were secreted as homogeneously glycosylated proteins that contained high mannose glycans. Furthermore, observation of GP1 ectodomain shedding from cells expressing wild type LASV GPC represents a novel aspect of arenaviral glycoprotein expression.</p> <p>Conclusion</p> <p>These results implicate GP1 as a chaperone for the correct processing and shuttling of GP2 to the cell surface, and suggest that native GPC SP plays a role in this process. In the absence of GP1 and GPC SP the GP2 protein may be processed by an alternate pathway that produces heterogeneously glycosylated protein, or the polypeptide may not fully mature in the secretory cascade in mammalian cells. The expression constructs developed in these studies resulted in the generation and purification of soluble, uncoupled GP1 and GP2 proteins from mammalian cells with quasi-native properties. The observation of GP1 ectodomain shedding from cells expressing wild type LASV GPC establishes new correlates of disease progression and highlights potential opportunities for development of diagnostics targeting the early stages of Lassa fever.</p

Wheat Variety Yield Response to Nitrogen and Sulfur Rates During the 2019–2020 Growing Season

Early spring visual sulfur (S) deficiency symptoms are increasingly a concern for Kansas wheat growers, but the extent of yield limitation due to S deficiencies and its interaction with nitrogen (N) supply is not well quantified in this environment. Our objective was to evaluate the responses of three wheat varieties to the interaction of N and S rates. The experiment was conducted in four Kansas locations during the 2019–2020 winter wheat growing season: Ashland Bottoms, Argonia, Belleville, and Hutchinson. These locations were selected to provide a range in soil textures and organic matter content, as these variables might impact the crop’s response to the S rate. All results are discussed, but only those for Ashland Bottoms and Belleville, the most contrasting sites in terms of yield potential and soil organic matter content, are shown. Treatments were arranged as a complete factorial structure with a split-split-plot design. Variety was the whole plot, N was the sub-plot, and S was the sub-sub plot. Nitrogen rates were 50, 100, and 150% of the Kansas State University Soil Testing Lab recommendations for a 60 bushel per acre yield, and S rates were 0, 10, 20, and 40 pounds of S per acre. Wheat varieties evaluated were Zenda, SY Monument, and LCS Mint. Increasing N rates improved grain yield at all locations. The yield increase depended on the S rate at Ashland Bottoms (i.e., treatments not receiving S were non-responsive to N) but not at the remaining locations. Wheat varieties differed in grain yield at all locations regardless of N rate except for Argonia, where Zenda increased yields linearly with increases in N rate, whereas the remaining varieties showed a linear-plateau response. Increases in N rate also increased protein concentration at all locations, and this increase depended on S rate at three locations. Varieties differed in protein concentration at all locations, and this difference depended on the N rate in Argonia. Our results suggest that winter wheat response to the interaction between N and S fertilizer rates is location-specific, with greater chances of response in soils with sandier texture and lower organic matter contents

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

<p>Abstract</p> <p>Background</p> <p>There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2).</p> <p>Results</p> <p>Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP) fusions in the Rosetta strains of <it>Escherichia coli </it>(<it>E. coli</it>) using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC). Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF) against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA).</p> <p>Conclusion</p> <p>These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.</p

Genetic Variation and Breeding Potential of Phytate and Inorganic Phosphorus in a Maize Population

Seed P is predominantly bound in the organic compound phytate, which makes the bioavailability of P low for monogastric animals fed maize (Zea mays L.)-based diets. Decreasing phytate and increasing inorganic P (Pi, an available form of P) concentrations in maize grain would be desirable to help ameliorate environmental problems associated with high P in feces. Our objective was to investigate the potential of improving the P profile of maize grain through breeding and selection. Ninety S1 families from the BS31 population were evaluated at two locations for phytate, Pi, and other grain quality and agronomic traits. Phytate concentrations ranged from 1.98 to 2.46 g kg−1, and the broad-sense heritability (H) was relatively low (0.60). Both genetic variance and H (0.84) were much greater for Pi Few unfavorable genetic correlations were observed between either Pi or phytate and other key economic traits. Also, selection differentials of multiple trait indices indicated that the P profile of maize grain and grain yield and moisture could be improved simultaneously. Many cycles of selection will be needed, however, to reach desirable phytate and Pi concentrations, especially when selecting for multiple traits. Regardless, our results are encouraging given that the families evaluated were related S1 families and the number of families was relatively small

Development of a New Vaccine for the Prevention of Lassa Fever

BACKGROUND: Recent importation of Lassa fever into Germany, the Netherlands, the United Kingdom, and the United States by travelers on commercial airlines from Africa underscores the public health challenge of emerging viruses. Currently, there are no licensed vaccines for Lassa fever, and no experimental vaccine has completely protected nonhuman primates against a lethal challenge. METHODS AND FINDINGS: We developed a replication-competent vaccine against Lassa virus based on attenuated recombinant vesicular stomatitis virus vectors expressing the Lassa viral glycoprotein. A single intramuscular vaccination of the Lassa vaccine elicited a protective immune response in nonhuman primates against a lethal Lassa virus challenge. Vaccine shedding was not detected in the monkeys, and none of the animals developed fever or other symptoms of illness associated with vaccination. The Lassa vaccine induced strong humoral and cellular immune responses in the four vaccinated and challenged monkeys. Despite a transient Lassa viremia in vaccinated animals 7 d after challenge, the vaccinated animals showed no evidence of clinical disease. In contrast, the two control animals developed severe symptoms including rashes, facial edema, and elevated liver enzymes, and ultimately succumbed to the Lassa infection. CONCLUSION: Our data suggest that the Lassa vaccine candidate based on recombinant vesicular stomatitis virus is safe and highly efficacious in a relevant animal model that faithfully reproduces human disease

Genetic mapping of quantitative trait loci for end-use quality and grain minerals in hard red winter wheat

To meet the demands of different wheat-based food products, traits related to end-use quality become indispensable components in wheat improvement. Thus, markers associated with these traits are valuable for the timely evaluation of protein content, kernel physical characteristics, and rheological properties. Hereunder, we report the mapping results of quantitative trait loci (QTLs) linked to end-use quality traits. We used a dense genetic map with 5199 SNPs from a 90K array based on a recombinant inbred line (RIL) population derived from ‘CO960293-2’/‘TAM 111’. The population was evaluated for flour protein concentration, kernel characteristics, dough rheological properties, and grain mineral concentrations. An inclusive composite interval mapping model for individual and across-environment QTL analyses revealed 22 consistent QTLs identified in two or more environments. Chromosomes 1A, 1B, and 1D had clustered QTLs associated with rheological parameters. Glu-D1 loci from CO960293-2 and either low-molecular-weight glutenin subunits or gliadin loci on 1A, 1B, and 1D influenced dough mixing properties substantially, with up to 34.2% of the total phenotypic variation explained (PVE). A total of five QTLs associated with grain Cd, Co, and Mo concentrations were identified on 3B, 5A, and 7B, explaining up to 11.6% of PVE. The results provide important genetic resources towards understanding the genetic bases of end-use quality traits. Information about the novel and consistent QTLs provided solid foundations for further characterization and marker designing to assist selections for end-use quality improvements.Horticulture and Landscape Architectur